Penicillium oxalicum GZ-2, but in Trichoderma reesei RUT-C30, cellulose induces a more efficient response. To understand the reasons, we designed an artificially simulated plant biomass (cellulose plus xylan) to study the roles and relationships of each component in the production of lignocellulolytic enzymes by P. oxalicum GZ-2.Agricultural residue is more efficient than purified cellulose at inducing lignocellulolytic enzyme production in egl1, egl2, egl3, sow, and cbh2) and hemicellulase genes (xyl3 and xyl4) were significantly upregulated (P <0.05). The proportion of biomass-degrading proteins in the secretome was altered; in particular, the percentage of cellulases and hemicellulases was increased. The percentage of cellulases and hemicellulases in the AX secretome increased from 4.5% and 7.6% to 10.3% and 21.8%, respectively, compared to the secretome of the A treatment. Cellobiohydrolase II (encoded by cbh2) and xylanase II (encoded by xyl2) were the main proteins in the secretome, and their corresponding genes (cbh2 and xyl2) were transcripted at the highest levels among the cellulolytic and xylanolytic genes. Several important proteins such as swollenin, cellobiohydrolase, and endo-beta-1,4-xylanase were only induced by AX. Bray-Curtis similarity indices, a dendrogram analysis, and a diversity index all demonstrated that the secretome produced by P. oxalicum GZ-2 depended on the substrate and that strain GZ-2 directionally adjusted the compositions of lignocellulolytic enzymes in its secretome to preferably degrade a complex substrate.The changes in lignocellulolytic enzyme activity, gene expression involving (hemi)cellulolytic enzymes, and the secretome of cultures grown on Avicel (A), xylan (X), or a mixture of both (AX) were studied. The addition of xylan to the cellulose culture did not affect fungal growth but significantly increased the activity of cellulase and hemicellulase. In the AX treatment, the transcripts of cellulase genes  contains supplementary material, which is available to authorized users. With the exhaustion of fossil fuels and the increasing global demand for fuel, the enzymatic conversion of lignocellulosic feedstocks into fermentable sugars has become an attractive alternative for clean and sustainable fuel production. Fungi are the major sources of lignocellulolytic enzymes . The ligTrichoderma spp., Aspergillus spp., and Penicillium spp. Cellulose itself cannot directly trigger the induction of lignocellulolytic enzymes because it is insoluble [Trichoderma reesei [Trichoderma longibrachiatum cultured on a mixture of lactose (0.8%) and xylan (0.2%) was found to result in significantly higher levels of both xylanase and cellulase than did cultures on either substrate alone [Over the past decades, many high-production cellulolytic fungi have been isolated and reported ,4. Howevnsoluble . Solublea reesei -9. Hemica reesei . Trichodte alone . These rTrichoderma and Aspergillus have been well studied and characterized [Aspergillus niger [Trichoderma spp. and Aspergillus spp. induced by different types of plant biomass [P. oxalicum GZ-2. However, this feature did not exist in strain T. reesei RUT-C30 (data not published in our lab), suggesting that P. oxalicum GZ-2 possibly has different regulation and induction mechanisms for producing lignocellulolytic enzymes.Agricultural wastes are renewable and abundant worldwide, and they are available for use as cheap feedstock to produce biomass-degrading enzymes. Applications of these enzymes to saccharify plant biomass for the production of bioethanol have been reported elsewhere -14. Manycterized -19. Receus niger -22. Many biomass ,23-26. H biomass . In our P. oxalicum GZ-2 responds to plant biomass and induces an enzyme cocktail to degrade plant polymers may result in new strategies to improve the production of second-generation biofuels. The relationships and roles of each complex plant biomass component (such as cellulose and xylan) in inducing and regulating lignocellulose-degrading enzyme gene expression and protein profiles are still poorly understood. Therefore, an artificially mixed substrate containing a mixture of Avicel and xylan was designed to simulate plant biomass in this study. The objective of this work was to investigate how inducible hydrolytic enzymes respond to cellulose and xylan and their relationships and roles in protein expression along with the activity of lignocellulolytic enzymes.Understanding how the filamentous fungus P. oxalicum GZ-2 was evaluated in a time course when glucose (G), Avicel (A), xylan (X), and a mixture of Avicel and xylan (AX) were used as the sole carbon source, and the results are shown in Figure T. reesei RUT-C30 are shown in Additional file The production of various enzymes by P. oxalicum GZ-2, we examined the growth behavior of P. oxalicum GZ-2 on various substrates. As shown in Additional file P. oxalicum GZ-2 on AX occurred at a slightly faster rate but with no significant difference before the third day. The fungal cells on A always increased with time and peaked at the end of the fermentation. The protein concentrations in the four substrates were determined and are shown in Additional file To determine whether the increase in enzymatic activity by AX was due to an increase in fungal biomass from The protein profile by SDS-PAGE is shown in Figure P. oxalicum GZ-2 under different substrate treatments are shown in Figure cbh2 transcript was significantly higher than those of the other enzyme-encoding genes in all the treatments. Similarly, significantly higher transcript levels of egl1, egl2, egl3, sow, and cbh2 were detected in the AX treatment than in the A or X treatment (P <0.01). Unexpectedly, the highest level of the bgl transcript was observed in the X treatment. The expression level of cbh1 was maintained at a low level in all treatments. A very low level of constitutive expression was observed for all the genes when G was used as a substrate.The transcript levels of lignocellulose-degrading genes in xyl1, xyl2, xyl3, xyl4, arf, b-x, and ma) at various levels . Interestingly, the transcript levels of the genes xyl3 and xyl4 were induced significantly more by AX than by the other substrates (at least P <0.05). No transcript level difference of xyl1 existed between the X and AX treatments, but it was significantly higher than that in the A treatment (P <0.01). The expression levels of arf and b-x were induced significantly more by X than by the other substrates. However, it is noteworthy that A was the substrate that most strongly induced the expression of ma.All substrates  induced the expression of all the hemicellulolytic genes  are listed in Additional file P. oxalicum GZ-2 were further evaluated using the Shannon-Wiener index (H) and the Simpson diversity index (D)  was used to analyze and identify the secretomes induced by different substrates. In the Venn diagram Figure , 108 of A total of 101 proteins were identified when GZ-2 was grown in a medium containing 1% (w/v) G, and 26 proteins were only induced by G. Only one cellulase  was identified in the G culture. In the secretome obtained from the A culture, 157 proteins were identified, and 43 proteins were only induced by A. Three major types of cellulase  and many hemicellulases were present in this secretome. A total of 141 proteins were identified, and 24 proteins exclusively existed in the X secretome. In the secretome of AX, 86 proteins were identified, 15 of which were only induced by AX. Two cellulases (swollenin and cellobiohydrolase) and four hemicellulases were specifically induced by AX.P. oxalicum GZ-2 was grown on G. The comparative quantitative expression abundances of the cellulolytic proteins on the different substrates are presented in Table Complete cellulose degradation requires three major cellulases: endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, and beta-glucosidase. The greatest number of cellulose-degrading enzymes (nine cellulases) including three major cellulases, swollenin, and cellulose monooxygenase (Cel61A) were identified in the AX secretome. The proportion of cellulases in the AX secretome 10.3%) was more than twofold greater than that of the A (4.5%) or X (5.0%) secretome. The same number of cellulases (seven) was identified in the A and X secretomes. Only one cellulase was detected when % was morP. oxalicum GZ-2 was grown on polymeric substrates  endo-beta-1,4-xylanase and GH3 beta-xylosidase are constitutively expressed using G as a substrate. The comparative expression abundances of the hemicellulolytic proteins cultured with various substrates are presented in Table P. oxalicum GZ-2 was cultured with AX compared to X, and the AX/X ratios were 33.5, 1.4, 3.4, 2.4, and 4.9, respectively. Many other glycoside hydrolases involved in the hydrolysis of glycosidic bonds were identified in the GZ-2 secretome. Although no starch is present in any of the substrates, amylase and glucoamylase were identified in the A, X, and AX secretomes.The degradation of complex hemicelluloses requires several synergistic actions of different hemicellulases including xylanases, beta-xylosidase, beta-1,4-mannanase, acetyl xylan esterase, and alpha-L-arabinofuranosidase. These enzymes were present when This study identified nine pectin-degrading enzymes and four chitin-hydrolyzing enzymes  more efficiently than purified cellulose to produce cellulase. In contrast, T. reesei RUT-C30 was found to be more effective at secreting cellulolytic enzymes with purified cellulose as an inducer (data not published). As we know, cellulose is one of the most suitable substrates to induce cellulases for many fungi, especially for T. reesei RUT-C30 [P. oxalicum GZ-2 were different from those of T. reesei RUT-C30. The reasons why agricultural waste induces more enzymes for P. oxalicum GZ-2 are unclear. We hypothesize that xylan as the major component in the hemicellulose synergizes with cellulose to enhance lignocellulolytic enzyme induction. To validate this hypothesis and further evaluate the influence of the composition of complex substrates on enzyme induction, an artificial substrate containing a mixture of Avicel and xylan was designed to simulate plant biomass to study these questions.In a previous study, we found that  RUT-C30 ,28. ThisP. oxalicum GZ-2 by the mixture of cellulose and xylan was significantly higher than that induced by purified cellulose. However, few reports have sought to explain why a mixture of xylan and cellulose enhances cellulase production. Contrary to T. reesei RUT-C30, purified cellulose is a poor inducer of cellulase production for P. oxalicum GZ-2. The result suggested that a difference potentially exists between these two strains in their production and regulation of cellulases. Another possible explanation is that T. reesei RUT-C30 is a mutant strain lacking repression by glucose, whereas P. oxalicum GZ-2 is not. In the A culture supernatant of strain GZ-2, a low concentration of glucose was detected during the fermentation period, which can repress the expression of cellulase. Wei et al. reported that a catabolite-repression-resistant mutant strain (Penicillium decumbens JU-A10) has a higher secretion capacity for cellulolytic enzymes than the wild-type strain Penicillium decumbens 114-2 [T. reesei RUT-C30 produces cellulolytic enzymes more efficiently on cellulose medium.As reported previously , the celP <0.05), whereas the biomass of the A treatment was increased gradually and peaked at the end of fermentation. This may have happened because the low concentration reducing sugar was constantly supplemented as nutrition for growth from the enzymatic hydrolysis of A (data not shown). These results suggested that the increase in lignocellulolytic enzyme activity was not caused by better growth with the mixed carbon source.The growth behavior of strain GZ-2 with various substrates was determined to evaluate the effect of fungal growth on enzymatic activity. The fungal cells of strain GZ-2 grew on the AX medium slightly faster than on the other substrates during the first three days (no significant difference at Trichoderma reesei (xyr1) [Aspergillus niger (XlnR) [XlnR plays a key role in the xylan-triggered induction of cellulase and hemicellulase. For example, D-xylose and cellobiose trigger XlnR-dependent expression of xylanolytic and cellulolytic genes in A. oryzae [The CMCase activity significantly increased when xylan was added to the cellulose medium; however, the culture supernatant of X did not display any CMCase activity. It was obvious that the increased CMCase activity was not due to xylan induction. We conjecture that xylan or its derivative products may activate regulatory factors that are able to enhance cellulase and hemicellulase expression. The xylanase regulator has been identified in i (xyr1) ,32 and Ar (XlnR) ,34 as th. oryzae . Low cel. oryzae -39.egl1, egl2, egl3, sow, and cbh2) were strongly expressed when AX was used as the substrate, explaining the high level of cellulase activity in the culture supernatant of AX. As we know, biomass-degrading enzymes are mainly regulated at the transcript level [egl1, egl2, and egl3 were measured in the AX treatment, indicating that these enzymes were major proteins in the control of CMCase activity. The expression level of cbh2 is remarkably high compared with that of other genes. Consistent with the transcript result, the most abundant protein band (C3) observed on SDS-PAGE  was also the major cellulase in the strain GZ-2. The transcript level of cbh2 was significantly higher in the AX culture among the four substrates, suggesting that the cellobiohydrolase II was strongly induced by AX. In most fungi, including T. reesei and P. oxalicum, cellobiohydrolase is the main cellulase in the cellulolytic system [bgl and the bright hydrolysis zone in-gel activity detection. Jørgensen et al. reported a similar result in which oat spelt xylan induced more beta-glucosidase than cellulose for Penicillium persicinum IBT 13226 [Results of the quantitative polymerase chain reaction (q-PCR) for cellulolytic genes clearly showed that most genes cellulolytic enzymes, zymogram gels incorporated with sodium carboxymethyl cellulose (CMC-Na) or beechwood xylan were run. Zymography is a powerful technology compared to the traditional colorimetric method because it not only can measure enzymatic activity but also visualize hydrolytic enzymes . Using zs Avicel . The sams Avicel .et al. reported that the addition of xylan to cellulose medium significantly increased xylanase activity and GH10 xylanase production in the basidiomycete Phanerochaete chrysosporium [T. reesei RUT-C30  induced by AX suggest that two xylanases (encoded by xyl3 and xyl4) are the reason for the increase in xylanase activity in the AX culture. The protein band (X10) showed the maximal hydrolysis zone . The high xylanase II abundance is in agreement with its gene (xyl2) being strongly expressed when X is used as a substrate. Although the content of GH11 xylanase II (encoded by xyl2) was dominant in the X secretome, the specific activity of xylanase II was found to be weaker than that of the other two xylanases (encoded by xyl3 and xyl4) [xyl3 and xyl4) are the key enzymes contributing to the high xylanase activity instead of xylanase II when cellulose is added to xylan medium. These results from the xylanase zymogram further confirmed that the addition of cellulose to xylan increased the abundance of hemicellulase species. Xylanase activity could be detected by neither colorimetry nor zymography when strain GZ-2 was grown on G, indicating the repression of these enzymes by glucose.It is well known that xylanase is usually induced by xylan polymers and that cellulose is a poor substrate for producing xylanase. For example, Hori osporium . As expend xyl4) . These ret al., in which the expansin-like protein (like the swollenin protein) only existed in the surgarcane bagasse secretome among four secretomes  of Trichoderma harzianum [Postia placenta [Aspergillus fumigatus [Trichoderma harzianum [P. oxalicum GZ-2 strictly depended on the substrate and that strain GZ-2 directionally changed the proportion of lignocellulolytic enzymes in its secretome according to the component of the substrate to promote subsistence on the complex substrate. Hori et al. studied the effects of xylan on the secretome of the basidiomycete Phanerochaete chrysosporium cultivated on cellulose medium using two-dimensional electrophoresis [P. chrysosporium. Furthermore, these authors’ studies are mainly focused on xylan-degrading enzymes, but less work was conducted to investigate the cellulase effect. Gómez-Mendoza et al. comparatively studied the secretomes of Trichoderma harzianum on CMC, xylan, and sugarcane bagasse [Trichoderma harzianum displayed the highest cellulolytic and xylanolytic activities, it did not correspond to greater proteome complexity because the cellulose-induced secretome was even more diverse.Interestingly, the secretome was significantly altered by the addition of xylan to the cellulose medium. In particular, the proportion of cellulases and hemicellulases was almost twofold higher in the AX secretome than in the others, which may be an important reason why lignocellulolytic enzyme production was enhanced when AX was used as the substrate. Three major cellulases, namely endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, and beta-glucosidase, were also expressed in the A and X secretomes. However, the swollenin protein (525580909), which disrupts crystalline cellulose to enhance cellulosic substrate hydrolysis, and GH7 cellobiohydrolase (525584244) were only present in the AX secretome. A similar result was reported by Gómez-Mendoza arzianum . It is nplacenta , Aspergiumigatus , and Triarzianum . The putphoresis . These a bagasse . Althouget al. identified six genetically different endo-beta-1,4-xylanases in the Penicillium decumbens 114-2 secretome induced by a mixed medium (CW) consisting of cellulose plus wheat bran [P. decumbens 114-2 secretome of CW, although five genes were detected in the transcriptome. In this study, two beta-xylosidases were detected in each of the A, X, and AX secretomes, whereas only one was induced by G. These results suggest that P. oxalicum GZ-2 has more advantages in the degradation of hemicelluloses than does P. decumbens 114-2. Thus, many biomass-degrading enzymes are identified in the secretome, suggesting the potential of P. oxalicum GZ-2 as a versatile cell factory for the production of extracellular enzymes. Further works on molecular regulations are needed in order to understand the mechanisms of lignocellulolytic enzyme production under the induction of complex substrates.Many studies have demonstrated that xylan is an effective inducer of hemicellulases ,47,49,50eat bran . Beta-xyP. oxalicum GZ-2 strictly depended on the nature and component of the substrate. The fungus GZ-2 changed the proportion of lignocellulolytic enzymes in its secretome according to the type of substrate. These results were able to successfully explain the more highly efficient enzyme production induced by a complex substrate.In lignocellulolytic enzyme production, cellulose and xylan have positively synergistic effects and they play an important role in the induction of highly efficient lignocellulolytic enzymes. More diverse functional protein species and the higher expression of certain enzymes are the primary contributors to the positively synergistic effect that results in greater enzymatic activity. The composition of the secretome of P. oxalicum GZ-2 used in this study was isolated and identified as previously reported [T. reesei RUT-C30 (ATCC 56765) was kindly provided by Irina S. Druzhinina . The two strains were grown on potato dextrose medium at 30°C for 6 to 7 days. Conidia were harvested and made into a suspension at a concentration of 1 × 107 conidia/mL. For secretome production, a basal medium 2SO4, 1.0 g of tryptone, 0.3 g of urea, 0.4 g of CaCl2.2H2O, 0.3 g of MgSO4.7H2O, 7.5 mg of FeSO4.7H2O, 2.0 mg of MnSO4.H2O, 2.0 mg of ZnSO4, and 3.0 mg of CoCl2 in 1,000 mL of water, pH 5.0) was supplemented with 1% (w/v) cellulose , xylan , a mixture of cellulose and xylan , or glucose (treatment G). The ratio of A:X =2:1 was chosen to reflect the cellulose and hemicellulose composition of the majority of agricultural residues. An aliquot of 100 mL of supplemented medium was placed in a 500-mL Erlenmeyer flask, sterilized at 115°C for 30 min, inoculated with 1 mL of conidia suspension (1 × 107 conidia/mL), and incubated at 30°C for 7 days (170 rpm). Each treatment was replicated three times. The fermented broths were centrifuged at 12,587 × g at 4°C for 20 min, the precipitated material was discarded, and the supernatant was filtered through a 0.22-μm membrane and further concentrated by freeze-drying. One hundred micrograms of the dried powder was dissolved in 10 mL of 50 mM Tris-HCl buffer (pH 8.0) and ultrafiltered through a 10-kDa molecular weight cut-off (MWCO) membrane . The total protein content in the ultrafiltered solution was determined using a MicroBCA protein assay kit  with bovine serum albumin as the standard. The protein solution was stored at -80°C for later proteomic analysis.The reported  and has et al. [During fermentation, 2-mL samples were collected at regular intervals for lignocellulolytic enzyme activity assays. The filter paper (FPase) and endoglucanase (CMCase) activities were determined as described by Ghose  using Whet al. . The redet al.  with glup-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, and p-nitrophenyl beta-D-cellobioside as substrates according to Parry et al. [2CO3. A Multi-detection Microplate Reader  was used to read the absorbance at 405 nm. One unit of activity was defined as the amount of enzyme that was required to release 1 μmol of nitrophenol per minute.The beta-glucosidase, beta-xylosidase, and cellobiohydrolase activities were measured using 10 mM y et al. . The reaA separate fermentation experiment was conducted to determine the fungal biomass. The entire fermented suspension was collected (X and G), filtered through dried filter paper, washed with MilliQ water  three times, and dried at 75°C to constant weight. The mycelial weight (A or AX) was calculated as the difference between the total dry weight of the solids  and that of the residual cellulose. The content of residual cellulose was determined according to Ahamed and Vermette .et al. [et al. [Penicillium oxalicum 114-2 that was downloaded from the National Center for Biotechnology Information (NCBI) database [P <0.05.Proteins in the ultrafiltered solution were profiled using SDS-PAGE (11% (w/v) polyacrylamide gel with a 5% stacking gel) as described by Laemmli  by loadiet al. . To do tet al. . Both th [et al. . The digdatabase  using MaP. oxalicum GZ-2 induced under different carbon sources at 30°C for 48 h were ground in liquid nitrogen and suspended in Trizol reagent . The total RNA was extracted following the manufacturer’s protocol. Reverse transcription (RT) was performed using the PrimeScript™ RT reagent Kit and the gDNA Eraser Kit . The RNA concentration was determined at 260 nm using a NanoDrop ND-2000 .For total RNA extraction, fresh mycelia of egl1, KF233750; egl2, KF233751; and egl3, KF233752), one beta-glucosidase gene , one swollenin gene , and two cellobiohydrolase genes . Seven known genes encoding hemicellulose-degrading enzymes were selected, including four xylanase genes , one beta-xylosidase gene , one alpha-L-arabinofuranosidase gene , and one beta-1-4-mannanase gene . The gene expression copy number was calculated using a standard curve for each gene as described by Lee et al. [To determine the expression of lignocellulose-degrading genes, real-time quantitative PCR (q-PCR) was performed using the primers listed in Additional file e et al. . The tra4HCO2 (50 mM) (pH 7.8) at 37°C for 18 h. The peptide mixtures after digestion were lyophilized, desalted by Empore C18-SD disk cartridge  and further dried in a vacuum centrifuge.Equal amounts of protein (2.0 mg) from each experimental condition were denatured in 1 mL of 8 M urea and reduced in 5 mM dithiothreitol (DTT) in 50 mM Tris-HCl (pH 8.0) at 95°C for 20 min. After cooling to room temperature, the protein was alkylated in 25 mM iodoacetamide (IAA) for 45 min in the darkness at room temperature. The final products were digested by adding 2% sequencing-grade trypsin  in urea (1.0 M)-NHThe dried peptides were resuspended in 200 μL of 0.1% formic acid and separated by an Acclaim PepMap 100 column   capillary with a 15-cm bed length using an UltiMate 3000 nano-HPLC  at a flow rate of 300 nL/min linked to an LTQ Orbitrap XL mass spectrometer . Two solvents, A (0.1% formic acid) and B (aqueous 80% acetonitrile in 0.08% formic acid), were used to elute the peptides from the nanocolumn. The gradient elutions were achieved using 5 to 40% of solvent B for 32 min, 40 to 95% B for 19 min, and maintained at 95% B for 9 min, with a total run time of 60 min. The electrospray voltage and the temperature of the ion transfer capillary were 2.2 kV and 200°C, respectively. The LTQ Orbitrap XL mass spectrometer was run in data-dependent acquisition mode using Xcalibur 2.2 software (Thermo Scientific) using the positive ion mode for data acquisition. Full-scan MS spectra (from m/z 350 to 1800) were acquired in the Orbitrap with a resolution of 60,000. The 10 most intense precursor ions greater than the threshold of 500 counts were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was employed within 60 s to prevent the repetitive selection of peptides. A total of four technical replications were obtained for each biological replicate.Penicillium oxalicum 114-2 that was downloaded from the NCBI database [All the MS/MS spectra were matched to specific proteins by searching against the FASTA proteome database of database  using Prdatabase . The moldatabase  accordinet al. [For the quantitative analysis, SIEVE , a commercial label-free quantification package, was used to compare the relative abundance of proteins between the different carbon source treatments. Four raw MS files from each treatment were analyzed using the SIEVE software according to Katz et al. . The expP ≤0.05 or 0.01). All of the statistical analyses were performed using SPSS version 19.0  and Microsoft office excel 2010. The LC-MS/MS alignment results are listed in Additional file The experiments were carried out in triplicate, and the results were subjected to Tukey’s HSD test for three independent samples at a 5% or 1% level of significance (


A total of 26 complete 1.2-cm bone defects were created in the ulna of rabbits, which were treated with CS, simvastatin-loaded CS or recombinant human bone morphogenetic protein 2 (rhBMP)-2-loaded CS. Simvastatin was highly efficiently released from simvastatin-loaded CS at the onset and stable release was maintained. Alkaline phosphatase was highly expressed in the MSCs co-cultured with simvastatin/CS scaffolds for 7 and 14 days. The defects treated with rhBMP-2-loaded CS and simvastatin-loaded CS showed significantly higher X-ray analysis scores and a larger amount of bone formation as determined by histology compared with the CS group (P<0.05). No significant differences in the X-ray score and bone formation were observed between groups with rhBMP-2-loaded CS and simvastatin-loaded CS (P>0.05). Simvastatin is capable of promoting osteogenic differentiation of MSCs in vitro and stimulating bone regeneration when locally released from CS scaffolds into bone defects. The beneficial effect of simvastatin was similar to that of rhBMP-2. In conclusion, the present study suggested that the simvastatin-loaded CS scaffolds may have great potential in bone tissue engineering.A number of clinical and experimental studies have investigated the effect of simvastatin on bone regeneration. In the present study, the release of simvastatin from simvastatin-loaded calcium sulphate (CS) scaffolds and the effect of these scaffolds on osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs)  The repair of large bone defects, caused by injury, eradicated tumor masses or progressive periodontal diseases is challenging. Autologous bone grafts, allografts and alloplasts have been used for bone repair . AutologAmong the numerous bone substitutes, calcium-sulfate (CS) is safe, highly biocompatible, bioresorbable and osteoconductive ,7. In adin vitro and the effects of simvastatin-loaded CS on the regeneration of segmental bone defects in the ulna of rabbits were investigated.Thus, simvastatin-loaded CS may be attractive as a novel bone substitute enhancing bone regeneration. In the present study, the release of simvastatin from simvastatin-loaded CS scaffolds, the effect of simvastatin on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) ® , a medical-grade CS powder, was used in the present study. Simvastatin  was dissolved in 75% ethanol at a concentration of 100 mg/ml. rhBMP-2  was dissolved in phosphate-buffered saline  at the concentration of 1 mg/ml. For the preparation of simvastatin-loaded and rhBMP-2-loaded CS scaffolds, 0.48 g CS powder, 0.145 ml distilled water and 5 μl simvastatin solution or 0.48 g CS powder, 0.14 ml distilled water and 10 μl rhBMP-2 solution were aseptically mixed in a dish. The mixture was transferred into a circular mold of 4-mm diameter and 12-mm thickness to create cylinders for implantation. In addition, to compare the differences in simvastatin release between CS scaffolds of different weights at the same dose, 0.16 g CS powder, 0.045 ml distilled water and 5 μl simvastatin solution were mixed, then simvastatin-loaded CS scaffolds (0.16 g) of the same diameter were fabricated. Finally, 0.5 mg simvastatin was added to each scaffold.OsteosetThe simvastatin-loaded CS scaffolds  were placed in 5 ml PBS (Gibco-BRL) at 37°C and the PBS was changed at 1, 3, 4, 6, 8, 11, 14 or 21 days, respectively. At every time-point, the solution absorbance was measured at a wavelength of 238 nm using an ultraviolet-visible spectrophotometer, while the simvastatin concentration was determined from a standard curve prepared with various amounts of simvastatin.2 and the medium was changed every two days. When adherent cells reached 80–90% confluency, they were detached with 0.25% trypsin-EDTA (Gibco-BRL) and replated at a ratio of 1:3 in regular growth medium to allow for continued passaging. Only passage three cultures were used for the experiments.Rabbits heparinized bone marrow (BM) cells were aspirated from the humerus with an 18-gauge needle. The mononuclear cells were centrifuged at 1,000 × g for 10 min at room temperature. The cells were collected and resuspended in low glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum  and 1% antibiotics . Following 48 h in culture, the medium was removed and fresh medium was added to each flask. Cells were maintained at 37°C in a humid atmosphere with 5% CO5 cells/sample and cultured for 7 or 14 days in control medium. The medium was changed every two days. At 7 and 14 days, the medium was removed and the samples were transferred to a new plate, washed with PBS, followed by the addition of a cell lysis solution. The samples were then processed through two freeze-thaw cycles  to rupture the cell membrane and extract the proteins. ALP activity of the cell lysate was determined with a p-nitrophenyl phosphate (pNPP) phosphatase assay kit . Total protein content of the cell lysate was measured using a bicinchoninic acid (BCA) protein assay kit (Nanjing Jiancheng Bioengineering), according to the manufacturer’s instructions. Dividing the quantity of ALP by the amount of total protein normalized the specific amount of ALP. ALP activities of MSCs cultured on CS scaffolds were used as controls.The osteogenic differentiation of MSCs cultured with simvastatin-loaded CS scaffolds was examined by measuring alkaline phosphatase (ALP) activity expressed by the cells. Scaffolds were first placed in a six-well plate and MSCs were seeded with samples at a density of 5×10A total of 18 New Zealand white rabbits  weighing 2.5–3.0 kg were used for the study. All animal experimental instructions were approved by the Animal Care and Use Committee of Zhejiang University  and followed the ‘Principles of Laboratory Animal Care’ , as well as specific national laws . The animals were anesthetized with an intravenous injection of 3% sodium pentobarbital (30 mg/kg). A total of 24 complete bone defects of 1.2 cm were created with a high-speed saw under irrigation with physiological saline and the periosteum was removed. The radius was left intact for mechanical stability.The 36 defects were randomized into 3 groups (n=6 in each group) and treated with the CS scaffold (group A), simvastatin-loaded CS scaffold (group B) or rhBMP-2-loaded CS scaffold . The rabbits of each group were sacrificed at 4 or 8 weeks following surgery.The status of bone repair and growth of callus were observed in samples removed through the original incision following animal sacrifice.Anterior and posterior radiographs of the bone defects were obtained to observe bone healing four and eight weeks following implantation. Bone formation was assessed in each group following instructions with triple blinding according to the Lane-Sandhu X-ray scores  Table ITable I.Samples were fixed with 10% paraformaldehyde, decalcified with formate-sodium formate and embedded with paraffin. Sagittal plane sections (7-μm thick) from the interface region of each implant were prepared and stained with hematoxylin and eosin (H&E), then examined under a light microscope .To quantitatively determine the amount of newly formed bone, the histological sections were statistically analyzed at four and eight weeks following implantation following the procedures described previously . Three pLane-Sandhu X-ray scores and newly formed bone areas were examined by one-way analysis of variance. Data analysis was performed using SPSS software . Fisher’s Least Significant Difference test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.in vitro release pattern of simvastatin from simvastatin-loaded CS is shown in The Specific ALP expression increased on simvastatin-loaded CS scaffolds between 7 and 14 days . At 7 daAll rabbits had normal diets and movement following surgery and survived until the scheduled date of sacrifice without any apparent complications.At four weeks, the majority of defects in group A was filled with fibrous tissue . In grouThe X-ray images of each group are shown in The bone formation scores were evaluated according to the Lane-Sandhu score standard. The X-ray scores increased with time and the score of group A was significantly lower compared with groups B and C at postoperative weeks four and eight (P<0.05). The scores of group C were slightly higher compared with group B. However, there was no significant difference between groups B and C both at four and eight weeks P>0.05; .At four weeks following implantation the CS was mostly degraded. In group A, small amounts of woven bone were observed to form in the extremities of the defects. The outsides of the defects were filled with fibrous tissue . In grouAt eight weeks, the two ends of the original ulna were united with the regenerated new bone. The CS was completely degraded. Compared with four weeks, the newly formed bone resembled normal cortical bone. In group A, dense woven bone formed in the extremities and inside the defect. However, the outside was not regenerated with bone and the marrow cavity was not recanalized . In grouThe area of newly formed bone in groups B and C was significantly higher compared with group A at four and eight weeks following implantation (P<0.05). No significant difference in the area of the newly formed bone was observed between groups B and C at each time-point .The present study demonstrated that simvastatin was highly efficiently released from simvastatin-loaded CS, promoted the osteogenic differentiation of MSCs and stimulated bone regeneration when it was locally released from CS scaffolds into bone defects. The beneficial effect of simvastatin, locally applied from CS scaffolds, was similar to those of rhBMP-2.In the present study, simvastatin was incorporated into CS scaffolds and was gradually released in a sustained manner. The release assay showed that the release efficiency correlated with the volume of simvastatin-loaded CS. Simvastatin-loaded CS (480 mg) showed a more efficient release of simvastatin on day 1 as compared with simvastatin-loaded CS (160 mg), which had a lower volume. On day 1, ~11.3% of the simvastatin was released from simvastatin-loaded CS (480 mg). However, from the simvastatin-loaded CS (160 mg), only 5.2% of the simvastatin was released. In addition, the release of simvastatin from simvastatin-loaded CS (480 mg) was extended over a longer time period as compared with the 160 mg sample. At day 21, 71% of simvastatin was released. In the simvastatin-loaded CS (160 mg), ~85% of the loaded simvastatin was released at day 14. The results suggested that the simvastatin-loaded CS (480 mg) may provide a longer period of simvastatin release, as well as a highly efficient release of simvastatin at the onset.in vitro culture and MSCs could not proliferate in a medium containing >2.5 μM simvastatin (A previous study suggested that the appropriate concentration of simvastatin was 0.5–1 μM for vastatin . Therefovastatin .In vivo, through the degradation of CS, simvastatin was persistently and locally released to induce bone formation. Local application has a therapeutic advantage by preventing systemic side-effects. Previous studies have investigated the effects of locally applied simvastatin. In these studies, the doses of simvastatin used have been variable: 2.2 mg (et al (The mechanism of sustained release of simvastatin occurs through degradation of CS. : 2.2 mg , 0.5 mg : 2.2 mg ,20; 0.1,: 2.2 mg  showed p: 2.2 mg , 0.5 mg g (et al  found thet al (Simvastatin could stimulate the BMP-2 expression in osteoblasts and inhibit the osteoclastic activity ,23. Furtet al , simvastet al .et al (Notably, the present study revealed that simvastatin stimulated bone formation later than rhBMP-2. This was observed from the quality of repaired bone tissue at four weeks following implantation. rhBMP-2 may directly stimulate the progenitor cells and osteoblasts; however, simvastatin needs to stimulate endogenous expression of BMP-2 first . Therefoet al , no seriet al . Thus, sIn conclusion, simvastatin may be efficiently released from simvastatin-loaded CS and induce osteogenic differentiation of MSCs. In addition, the advantages of simvastatin and desirable effects of rhBMP-2 on segmental bone repair were successfully combined with an efficient local application. Compared with rhBMP-2, simvastatin may be considered a cheap, well-tested drug with a beneficial side effect profile, and therefore, may be a promising substance in terms of bone regeneration. The simvastatin-loaded CS scaffold may have great potential in bone tissue engineering.


Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity.D values of all six mutant proteins were higher than that of RGD-hirudin.Recombinant RGD-hirudin and six mutant variants , designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KThese findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin. Hirudomedicinalis hirudin) has come into use over the past decade in in vitro blood research. It blocks the thrombin-mediated conversion of fibrinogen to fibrin during clot formation, but unlike heparin, it is a direct thrombin inhibitor (DTI) [A highly specific thrombin inhibitor derived from the leech (or (DTI)  that is or (DTI) ,3.1-Ile49) stabilized by three disulfide bonds with [3, Asp5) [A previous study has shown that the inhibitor is a small peptide  that binds to active thrombin and irreversibly inactivates it . It has nds with . This conds with  and thronds with ,12 indic3, Asp5) ,13 and t62 and Asp65). Asp53 was mutated to Gln53, Glu58 was mutated to Pro58, and Glu66 was added. These changes improve the hydrophobicity of the protein and allow the recombinant RGD-hirudin to interact more effectively with the fibrinogen recognition exosite of thrombin, resulting in a specific activity of 12,000 ATU/mg [Pichia pastoris. The design of the six mutations was based on molecular simulations. The Titration Testing Method and BIAcore® surface plasmon resonance analysis (SPR) were used to test their thrombin inhibition activity. Anti-platelet aggregation activity was tested by classic turbidity assays.The novel recombinant RGD-hirudin, which contains an Arg-Gly-Asp (RGD) adhesion site recognition sequence, is a bi-functional molecule based on the structure of wild-type hirudin variant 2 . In the 0 ATU/mg . Given tPichia pastoris cells carrying the RGD-hirudin gene (Mut+) and pPIC9k-RGD-hirudin plasmid were stored in our lab. Briefly, the RGD-hirudin gene was synthesized in the Key Laboratory of Molecular Medicine at Fudan University. cDNA encoding RGD-hirudin was cloned into the plasmid pPIC9K, and this expression vector was transformed into Pichia pastoris GS115. Vector integration into the Pichia pastoris chromosome was confirmed by PCR [Accelrys Discovery Studio  was used for homology modeling (MODELER)  and dockd by PCR ,19. DNA The amino acid sequence of RGD-hirudin was obtained in our lab. The NCBI protein BLAST program was used to search the Protein Data Bank (PDB) and was used to select a template structure for RGD-hirudin homology modeling. From the selected templates, a three-dimensional model of RGD-hirudin was obtained by homology modeling using the software package Discovery Studio 3.1. Constructed models were refined by performing an optimized geometry calculation of the mechanics using augmented CHARMM in Discovery Studio 3.1. The quality of refined models was assessed on the basis of both geometry and energy. The stereo-chemical properties of the models were investigated with a Ramachandran plot using PROCHECK .To assess the interaction between RGD-hirudin and thrombin, docking the modeled structure of RGD-hirudin with the crystal structure of thrombin (PDB ID: 4HTC) chain H&L was performed. ZDOCK tested the different docking sites by moving the ligand around the receptor. To obtain more accurate predictions, we specified the angular step size for the rotational sampling of the ligand orientations as 6, and the shape complementarity, desolvation and electrostatic energy terms were used in the initial-stage ranking of the docked protein poses. The docking parameters are set as follow: Input Receptor Protein: 4HTC: Thrombin, Input Ligand Protein: RGD-Hirudin, Angular Step Size: 6, ZRank: True, Clustering: Top poses: 2000, RMSD Cutoff: 6, Interface Cutoff: 9, Maximum Number of Clusters: 60.The Mutation Energy Calculation was used to evaluate the effect of single-point mutations on the binding affinity in the RGD-hirudin-thrombin docking complex. This protocol performs amino acid scanning mutagenesis on a set of residues (selected according to the docking results) by mutating each to alanine. The energy effect on the binding affinity of each mutation (Mutation Energy) was calculated as the difference in the binding free energy between the mutant and wild-type proteins. The binding free energy is defined as the difference in the free energy between the complexed and unbound state. All interaction energy terms were calculated by CHARM using a Generalized Born implicit solvent model; these terms contained empirically scaled contributions of van der Waals and electrostatic interactions and a non-polar solvation energy term. The mutation energy function contained also a side chain entropy term. The temperature was set at 37°C. This process was also implemented with the software package Discovery Studio 3.1.For preparation of six RGD-hirudin mutants, a Site-Directed Mutagenesis Kit was using two complementary primers which were described as follows Table  and the Pichia pastoris, then purified by gel filtration and anion exchange chromatography as previously described [600 of 5.8. Cells were harvested by centrifugation at 3000 rpm for 30 min, washed with 100 mM potassium phosphate (pH 6.0), and carefully resuspended in 200 mL sterile BMM. Samples were incubated at 30°C for another 72 h, with pulses of 0.5% (v/v) methanol added every 12 h. The culture was centrifuged and the supernatant was loaded onto a Sephacryl-S100 column (7.5 cm × 80 cm), pre-equilibrated with 20mMol/L phosphate buffer . A sample of 400 mL was eluted from the gel filtration column and loaded onto a Q-Sepharose FF column (2.6 cm × 20 cm), also pre-equilibrated with 20mMol/L PB (pH 7.4). The column was washed with 20mMol/L PB (pH 7.4), followed by a single linear gradient of 0–1.0Mol/L NaCl-PB buffer. RGD-hirudin was eluted at 0.25Mol/L NaCl-PB. The sample that showed anti-thrombin activity was collected and desalted with a Sephadex-G50 column (1.6 cm × 20 cm). Loading sample volumes were standardized to 5 mL. Protein concentration was measured with the Bradford assay. The desalted samples were lyophilized and stored at −80°C.Wild type and mutant variants of RGD-hirudin were expressed in escribed ,21. BrieProtein samples (wild type and mutant variants of RGD-hirudin) were analyzed by 15% SDS-PAGE. The anti-thrombin activity of RGD-hirudin and the mutant variants was tested by the Titration Testing Method, according to Markwardt . BrieflyThrombin was diluted in 5mMol/L sodium acetate (pH 5.0), and 2000 response units were immobilized via amine coupling to CM5 sensor chip flow chambers . Briefly, proteins were mixed with equal volumes of freshly prepared 100mMol/L N-hydroxysuccinimide and 400mMol/L N-ethyl-N’-(dimethylaminopropyl) carbodiimide, and capping of unreacted carboxymethyl sites was achieved by injection of 1.0Mol/L ethanolamine, pH 7.0. A flow chamber was subjected to the immobilization protocol. RGD-hirudin and the six mutant variants were sequentially diluted in running buffer , pH 7.5) and injected over the surfaces at different concentrations  at 30 μL/min.Binding was monitored with a BIAcore T100 instrument. Between experiments, the surfaces were strictly regenerated with multiple pulses of 2Mol/L NaCl and 1.5Mol/L glycine-HCl, pH 2.5, followed by an extensive wash procedure with running buffer.a) and dissociation (Kd) rate constants were determined using the BIAevaluation 4.1 software and its equation for 1:1 Langmuir binding. From these values, affinities (KD) were calculated.After x- and y-axis normalization of the data obtained, the blank bulk refraction curves from the control flow chamber of each injected concentration were subtracted. Binding curves were displayed, and the association   and the Analysis of the Ramachandran plot of the refined model showed that 96.9% of the residues lie in the most favorable regions Figure B, which 3 in the N-terminus of RGD-hirudin formed one hydrogen bond with Gly210 of thrombin. Further, in the C-terminus of RGD-hirudin, Ser50 formed two hydrogen bonds with Glu192 of thrombin, Gln53 formed one hydrogen bond with Leu40 of thrombin, Asp55 formed two hydrogen bonds with Arg73 of thrombin, and both Glu57 and Ile59 were hydrogen-bonded to Gln38 of thrombin  cutoff and the interface cutoff both set to 10 angstroms. For a pose to be included in a cluster, the RMSD cutoff was the maximal RMSD of the ligand interface from the cluster center, and the interface cutoff was the size of the interface region between receptor and ligand. Poses were then re-ranked with Van Der Waals, desolvation and electrostatics energy terms. The best docking poses were selected, based on a higher dock score combined with certain types of interactions between hirudin variant 2 and thrombin. We hypothesized that the interaction between RGD-hirudin and thrombin is similar to that of hirudin variant 2 and thrombin, and therefore favored this type of interaction. Finally, pose 24 was chosen as the best docking complex  of RGD-hirudin might participate in the interaction with thrombin.We performed amino acid scanning mutagenesis of RGD-hirudin-thrombin complexes, mutating to alanine each of six residues shown by the docking results to be important. We then calculated the differences in the free energy of binding between the wild-type and mutated structures. The results showed that the binding affinity of the six single-point mutations with thrombin were weaker (weighted mutation energy greater than zero) than that of wild-type RGD-hirudin with thrombin. The effect of each mutation was destabilizing Table , which f3, Ser50, Gln53, Asp55, Glu57, Ile59) were mutated to Ala, and the proteins were purified by gel filtration and anion exchange chromatography, as described in the Materials and Methods. The average yields of pure protein from 200 mL of fermentation culture were: RGD-hirudin, 46 mg; Y3A, 50 mg; S50A, 42 mg; Q53A, 47 mg; D55A, 40 mg; E57A, 38 mg; and I59A, 55 mg /mg; S50A, 16000 ATU/mg; Q53A, 14000 ATU/mg; D55A, 12000 ATU/mg; E57A, 11000 ATU/mg; and I59A, 9000 ATU/mg  was calculated from the determined Ka and Kd values  are important for binding to thrombin, as each mutant protein binds with lower affinity than wild-type RGD-hirudin.To further analyze the binding properties of the six mutant variants of RGD-hirudin with thrombin and to establish the affinity constant of the binding, surface plasmon resonance analysis was performed. The binding activity of RGD-hirudin was compared with those of the six mutants  in a competitive non-radioactive binding assay using thrombin as a receptor. The KM Figure A, whereaHirudomedicinalis) [Hirudin, an antithrombotic substance produced by the salivary glands of the medicinal leech  ,26, is tcinalis) .Pichia pastoris and subsequently purified. It may be a more effective agent than wild-type hirudin for anti-coagulation and anti-thrombosis in post-anastomosis surgery. We propose that RGD-hirudin will be able to replace heparin or wild-type hirudin and reduce bleeding rates, due to its bi-functional action at a much lower dosage than wild-type hirudin [In our laboratory, a new type of recombinant RGD-hirudin has been successfully cloned, expressed in the methylotrophic yeast  hirudin . However52 to Glu66), which is essential for the hirudin-thrombin interaction, had not yet been determined. The overall structure of RGD-hirudin (1–66) developed above used the crystal structure of chain I of hirudin variant 2 from the hirudin-thrombin complex (PDB ID: 4HTC) and the NMR solution structure of chain A of recombinant RGD-hirudin (PDB ID: 2JOO) as templates for docking with thrombin. Based on the docking model and the structural characteristics of the RGD-hirudin-thrombin complex, we find that the mechanism of interaction is consistent with that of native hirudin and thrombin. In this interaction, the globular N-terminal domains interact with the active site of thrombin, while the anionic C-terminal tails bind to exosite 1 on thrombin (the substrate-binding site) [3, Ser50, Gln53, Asp55, Glu57 and Ile59) of RGD-hirudin are hydrogen-bonded to thrombin, according to the results of simulations. To validate these residues as functional sites, mutant variants were constructed. Anti-thrombin activity assays and surface plasmon resonance analysis were also performed. Previous studies have indicated that the first three residues in the N-terminus of hirudin represent about 30% of the binding energy [3) and C-terminus  are both crucial for inhibiting the activity of thrombin, for binding near its active site and forming hydrogen bonds with exosite I.Although our lab has previously conducted a study on the structure of RGD-hirudin in solution (1–49) , the strng site) . Six resg energy  and the g energy . Mutageng energy . Our res56 engages in strong Van der Waals, edge-to-face interactions with thrombin Phe34, according to our docking complex (data not shown). Thus, other mechanisms of interacting and binding should be investigated in the future.In this study, we focused on hydrogen bonding interactions between RGD-hirudin and thrombin. However, hydrophobic interactions, Van der Waals interactions, and salt bridges might be also exist. For example, the phenyl ring of PheIn summary, The purpose of this study is that we found the binding site and active centre of RGD-Hirduin. In future, these results might be useful to provide a structural basis for the redesign of lower molecular weight antithrombin peptides which would be developed to the new oral drugs to fight thrombotic diseases.


Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection worldwide, little is known about the natural course of the bacterial load during infection. We investigated the natural course of the bacterial load in the interval between screening and returning for treatment in genital and anorectal CT-infections.Although CT-positive patients, visiting our STI-clinic in the Netherlands from June 2011–January 2014, provided a second urogenital and/or anorectal sample when returning for treatment . Patient-record provided data about the days between samples and the date of last unsafe sex. Included patients were ≥18 years old, HIV-negative and did not report antibiotic use in the study-interval. CT load was quantified using qPCR. CT load was log-transformed, and a CT load difference (Δ-CT load) of >1 log was deemed clinically relevant. Chi-square test compared load category distributions over time , between sample types.274 patients provided 296 paired samples. Majority of samples had a stable CT load in the interval T1-T2 . Load decreased in 17–41% of patients, while ±10% of patients showed an increase in CT load. No association between Δ-CT load and the interval T1-T2 was observed. Large variations can be seen in CT load at T1 and over time.The majority (±90%) of patients have a stable or decreasing CT load in the time interval between screening and returning for treatment. The number of days between sampling was not associated with change in CT load. In the first month after the last unsafe sex, only stable CT loads were seen. Our data seems to indicate that when most patients visit an STI-clinic, recommended 2 weeks after infection, the infection has already been established or is in its downward phase. Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection (STI) worldwide, with over 100 million people affected . The median number of days since the last unsafe sex was 90 days (range 7–565) for vaginal swabs and 62 days (range 3–380) for urines.The CT load curve for the first 366 days since the last unsafe sexual exposure , shows lA decrease in CT load was first seen after one month (30 days) since the last unsafe sexual exposure. No patients showed an (sharp) increase in CT load in the first 2 months after the last unsafe sexual exposure.Within 30 days after sexual exposure, CT load showed a median Δ-CT load of 0.53 log CT/ml (range -0.40–0.97), while the CT load after one month had a median Δ-CT load of -0.35 log CT/ml (range -2.58–4.06). Split by sample type, vaginal swabs had a median Δ-CT load of 0.53 log CT/ml (range -0.40–0.97) within 31 days, and -0.45 log CT/ml (range -2.58–4.06) after this time. Urines had a median Δ-CT load of 0.57 log CT/ml (range 0–0.90) within one month, and -0.45 log CT/ml (range -1.09–1.21) after one month.We assessed the natural course of the CT load in the interval between screening and returning for treatment in patients visiting an STI-clinic patients. This is the first study to present quantitative data on the natural course of CT infections over time, in both urogenital infections and anorectal infections in men and women.We demonstrated a stable CT load in the majority (47–73%) of patients in the time interval between screening and returning for treatment, which ranged in this study from 3–28 days. Depending on the sample type, load decreased in 17–41% of patients, while only ±10% of patients showed an increase in CT load. The number of days between T1-T2 did not correlate to the Δ-CT load, i.e. a longer time between sampling did not result in a larger difference in CT load. Furthermore, the time since last unsafe sexual exposure (to CT) was not clearly related to the course of CT load, with large variations over time.Little is known about what determines CT load in humans , and eveIn this light, our results seem to indicate that the majority of patients visit the STI-clinic when the infection has already been established or is in its downward phase. This is surprising, as most current guidelines state that patients should get tested at least 2weeks after the last unsafe sexual practices based on CT’s incubation period of 1–3 weeks , 28. SomApproximately 50% of CT-positives clear their urogenital infection within a year , 7. In tA limitation of our study is the lack of data about prior CT infections in our patients, however, diagnosed prior infections are only a proxy of the total amount of previous CT infections and therefore may confound these analyses . Anotheret al. [et al. [It is very difficult to speculate on implications of these results on reinfection rates, progression of disease and associated pathology. Much is still unknown about the clinical implications of the bacterial load, and its correlation to pathology. A low CT load has been associated with subsequent clearance in pharyngeal positive patients . In thiset al.  demonstret al. . Russel  [et al.  demonstr [et al. –40. FurtIn short, CT load is stable or decreasing in the vast majority of STI-clinic patients in the time interval between diagnosis and treatment. The time-interval between samples is unrelated to the change in load over time, requiring further research to clarify other factors at play. We found only stable CT loads within the first month after the las unsafe sex, which might reinforce recent guidelines that propose instant CT-testing after unsafe sexual practises.


UF-fractionation of EYP hydrolysate increased slightly the antioxidant activity in permeate fractions (720.5–867.8 μM TE·g−1 protein). However, ORAC values were increased by more than 3-fold in UF-fractions prepared from PPP hydrolysate, which were enriched in peptides with molecular weight lower than 5 kDa. These UF-fractions were characterized by their lower N/P atomic ratio and higher phosphorus content compared to the same UF-fractions obtained from EYP-TH. They also contained high amounts of His, Met, Leu, and Phe, which are recognized as antioxidant amino acids, but also high content in Lys and Arg which both represent target amino acids of trypsin used for the hydrolysis of PPP.The objective of the study was to compare the antioxidant activity of two distinct hydrolysates and their peptide fractions prepared by ultrafiltration (UF) using membranes with molecular weight cut-off of 5 and 1 kDa. The hydrolysates were a delipidated egg yolk protein concentrate (EYP) intensively hydrolyzed with a combination of two bacterial proteases, and a phosphoproteins (PPP) extract partially hydrolyzed with trypsin. Antioxidant activity, as determined by the oxygen radical absorbance capacity (ORAC) assay, was low for EYP and PPP hydrolysates with values of 613.1 and 489.2 μM TE·g HowevA large number of food protein-derived peptides have been shown to possess antioxidant properties . Some naEgg yolk proteins (EYP) have been found to possess antioxidant activity in a linoleate emulsion system  and phosDelipidated EYP is a by-product of lecithin extraction from egg yolk in the egg-processing industry. However, the delipidation process using ethanol and hexane for removal of phospholipids impairs protein functionality and delipidated EYP display reduced solubility properties . Enzymatin vitro against oxidative stress in an assay using human intestinal epithelial cells [et al. [Phosphopeptides having molecular masses between 1 and 3 kDa, obtained by tryptic hydrolysis of phosvitin, have been found to be effective for enhancing calcium binding capacity and to inhibit the formation of insoluble calcium phosphate ,22. Thesal cells . Jiang aal cells  have dev [et al.  developeet al. [Bacillus subtilis), showed antioxidant activity, supporting the view that delipidated EYP could be considered as a source of novel antioxidative peptides. These findings also suggest that biological activities of protein hydrolysates can be related to synergistic contributions of amino acid composition, sequence and molecular weight of peptides.More recently Young et al.  investigUF-membrane-based separations of enzymatic hydrolysates can achieve the removal of peptides from non-hydrolyzed proteins and proteolytic enzymes . In addiThe objective of this study was to evaluate the impact of UF-fractionation on the antioxidant activity of two different enzymatic hydrolysates, the first one prepared from EYP and extensively hydrolyzed with a combination of two bacterial proteases, and the second one produced from PPP and partially hydrolyzed using trypsin. Both hydrolysates were further fractionated by a two-step UF-process using membranes with low molecular weight cut-off (5 and 1 kDa) to obtain UF-fractions with distinctive composition. The antioxidant activity of the hydrolysates and their UF-fractions were then assessed using the oxygen radical absorbance capacity (ORAC) assay.2.2.1.Bacillus subtilis, EC 3.4.21.62, HBI Enzymes Inc., Osaka, Japan) and protease N  were kindly provided by Dr Mine's laboratory . Trypsin VI from bovine pancreas, containing 2800 U·mg−1 of trypsin activity (EC 3.4.21.4) and 490 U·mg−1 of chymotrypsin activity (EC 3.4.21.4), was donated by Neova Technologie Inc. . All other chemicals used in the experiments were of analytical grade.Defatted egg yolk protein concentrate was a gift from Taiyo Kagaku Co. Ltd. . Alcalase  of 6X and followed by a diafiltration (DF) step using 10 diavolumes (DV) of water. The final retentate, so-called PPP, was lyophilized and stored at −35 °C until use.Preparation of PPP from EYP was performed as described in Chay Pak Ting et al. . Briefly2.3.et al. [Enzymatic hydrolysis of dephosphorylated EYP solution was performed as described by Young et al. . BrieflyDephosphorylated PPP solution was hydrolyzed with trypsin as described by Jiang and Mine . The solBoth enzymatic hydrolysis reactions (EYP and PPP) were stopped by UF-separation using a polyethersulfone 10 kDa MWCO membrane to remove the enzymes and non-hydrolysed proteins. The permeates, so-called total hydrolysates (EYP-TH and PPP-TH), were lyophilized.2, Millipore Corp., Bedford, MA, USA) at a transmembrane pressure of 1.38 bar until a VCR of 5X was reached. The permeate collected in this first stage was further ultrafiltered using a regenerated cellulose 1 kDa-membrane coupon with an effective area of 0.09 m2, which was mounted on a SEPA ST system . UF was performed at a transmembrane pressure of 2.76 bars until a VCR of 4X was reached. This process yielded four fractions for each TH: a retentate enriched in peptides >5 kDa (UF5-R), a permeate mainly composed of peptides <5 kDa (UF5-P), a second retentate enriched in peptides >1 kDa (UF1-R), and a second permeate containing peptides <1 kDa. All retentates and permeates were freeze-dried and stored at −35 °C until further analysis.EYP-TH and PPP-TH were rehydrated in water  then fractionated consecutively through UF membranes having MWCO of 5 and 1 kDa . The fir2.4.The degree of hydrolysis  of EYP hydrolysate was determined in final powder by the ratio AN/TN, where AN is the amino-nitrogen content as determined using the formaldehyde titration method , and TN g for 10 min. The supernatant was decanted and passed through a Whatman No. 1 filter paper. Total nitrogen content was determined in filtrate and initial solution as described below. NSI value is expressed in percentage and represents the nitrogen content in filtrate divided by the nitrogen content in initial solution.The solubility of EYP-TH and PPP-TH was measured by the nitrogen solubility index (NSI) method  over theXL . A molecular weight calibration curve was prepared from the average retention time of protein/peptide standards ranging from 654 Da to 78 kDa.Molecular weight distribution of EYP-TH, PPP-TH and their UF-fractions were determined by gel permeation chromatography  using a The nitrogen content in samples was determined by the Dumas combustion method using a LECO FP-528 apparatus . Protein content was calculated from the total nitrogen content of samples using a protein conversion factor of 6.25.Phosphorus content was determined by the colorimetric molybdenum method of Allen  after acet al. [Amino acid composition was determined as Diaz et al.  after ac2.5.et al. [The ORAC assay was performed as described by Cao et al. . The dri2.5.Statistical differences between antioxidant activity of enzymatic hydrolysates and their respective UF-fractions were determined by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test using total hydrolysate (TH) as control sample. Statistical analysis was performed using GraphPad Prism software (version 5.04).3.3.2.Bacillus amyloliquefaciens) and Protamax 1.5 (from Bacillus licheniformis), two bacterial proteases with low specificity. However, the high solubility values obtained for our hydrolysates can also be explained by the UF-removing of non-hydrolysed proteins and enzymes at the end of the enzymatic reaction [reaction .UF-fractionation of EYP-TH led to little variations in protein (71.2–75.9%) and phosphorus (0.32–0.42%) contents of retentates and permeates fractions. The impact of UF-fractionation on protein and phosphorus contents was more pronounced for PPP-TH, but this can also be related to a more extended range of peptide molecular weights in this partial hydrolysate. Finally, the increased purity of phophopeptides in TH and UF-fractions was reflected by the decrease of N/P atomic ratio. For EYP, the lowest value was observed in UF1-R (60.1%) whereas for PPP, phophopeptides seemed to be concentrated in permeate fractions UF5-P (6.8%) and UF1-P (5.5%). These results thus suggested that phosphopeptides released during enzymatic hydrolysis of EYP and PPP varied in terms of molecular weight and seems to be shorter in PPP-TH.et al. [The molecular weight (MW) distribution profile of protein/peptide components in enzymatic hydrolysates (EYP-TH and PPP-TH) and their UF-fractions are presented in et al.  reportedet al. .The first UF-fractionation using the 5 kDa membrane removed a high proportion of large and intermediate MW peptides from PPP-TH whereas the other UF-fractions  obtained from both hydrolysates were relatively similar in terms of MW distribution profile of peptides.The amino acid compositions of EYP, PPP, their TH and UF-fractions are reported in As observed for phosphorus , Ser con3.2.p < 0.001) was observed for the permeate fraction obtained with the 5 kDa-membrane, for which ORAC value was 867.8 μM TE·g−1 protein compared to 613.1 μM TE·g−1 protein for the EYP-TH. The impact of UF-fractionation of PPP-TH on its antioxidant capacity was much more important, with ORAC values (1501.1–1886.8 μM TE·g−1 protein) more than 3 folds higher for all the UF-fractions comprising peptides with MW < 5 kDa compared to PPP-TH (489.2 μM TE·g−1 protein).These results thus suggest that phosphoproteins contained in egg yolk could be responsible for its antioxidant activity, which also seems to be related to the molecular weight of peptides contained in the fractions. This is in agreement with the findings of Jiang and Mine  who estaIn an attempt to establish a relationship between antioxidant activity of the different extracts  and theiThe higher antioxidant capacity measured UF-permeates fractions (1 kDa and 5 kDa) compared to retentates  may resuOverall, our data on amino acid composition and phosphorus content of the UF-fractions provide some evidence about the contribution of specific amino acids and phosphorus content to the antioxidant activity of PPP's. In addition, our results suggest that PPP UF-fractions containing peptides with molecular weight lower than 5 kDa  and having the highest phosphorus content may contain the bioactive peptides. However, the relative contribution of molecular weight, phosphorus content and amino acid profile to the antioxidant activity cannot be established from the present study.4.in vitro antioxidant activity of the UF-permeates was significantly higher than those of the non-fractionated hydrolysates. The findings from the present study suggest that tryptic hydrolysis of egg phosvitin combined to UF-fractionation of hydrolysate could provide new opportunities for the development of health-promoting ingredients.The


Seasonal asymmetry in the interannual variations in the daytime and nighttime climate in the Northern Hemisphere (NH) is well documented, but its consequences for vegetation activity remain poorly understood. Here, we investigate the interannual responses of vegetation activity to variations of seasonal mean daytime and nighttime climate in NH (>30 °N) during the past decades using remote sensing retrievals, FLUXNET and tree ring data. Despite a generally significant and positive response of vegetation activity to seasonal mean maximum temperature ( Similarectively .R > 0.30) between R > 0.30) with the WAI (for ~15–17% of the samples) in both spring and summer   during 1950–2008 (R > 0.30) with mean Both partial correlation and ridge regression analyses of the relationships among an  and  : The pern summer  and S3, d summer  and S2c.n RegAS)  and S10.in RegTH . At the 950–2008  and S1. d RegTA) . The  exR > 0.30, p < 0.10) in ~17.5% of these regions  . Coinciderate NH .Contrary to the ambiguous relationships between AVG and 950–2008 . Additio950–2008  and sele950–2008 .et al., 20131625The results of both the partial correlation and ridge regression analyses demonstrate that the AVG in the boreal NH is generally temperature-limited: a warmer mean daytime temperature  responded to the mean seasonal VDNC: the satellite-derived AVHRR/GIMMS Normalized Difference Vegetation Index 18http://www.ncdc.noaa.gov/data-access/paleoclimatology-data/datasets/tree-ring) based on four criteria: 1) intact records of latitude, longitude, elevation, species and sample depth; 2) the chronologies cover at least 1950–1998 or extend to more recent times ; 3) the sample depth for each site-year is greater than 10; and 4) the altitude of the sample site is less than the 90th percentile of the elevation distribution of the surrounding 0.5° × 0.5° region based on the GTOPO30 dataset  to match the regional vegetation growth patterns and avoid site-specific representations. Our research group provided another 13 standard chronologies from northern China. Our chronologies satisfy the four criteria listed above (see Table S4 for details).In total, 149 standard TRI chronologies from the mid- and high-latitude NH were used to investigate the interannual responses of tree growth to the seasonal VDNC. Standard TRI chronologies were built following the standard procedures of dendrochronologyhttp://www.cru.uea.ac.uk/). The seasonal (in detail see section of sensitivity test) mean http://www.esa-soilmoisture-cci.org/). These data were produced following the method described by Liu et al. (2011) and Liu et al. (2012)42Monthly mean maximum Spearman partial correlation and ridge regression were performed to investigate the responses of vegetation activity in the mid- and high-latitude NH to the mean seasonal VDNC. The interannual sensitivity of vegetation growth to IAV in the seasonal VDNC was evaluated using the ridge regression coefficients. Ridge regression has been demonstrated to be an effective method for addressing collinearity in multivariate regressionIn this study, we attempted to define the seasons  in a manner that is consistent across the mid- and high-latitude NH. Notably, three different definitions of seasons were used in our study to verify the robustness of our conclusions relative to the arbitrary definitions of vegetation growth seasons in the mid- and high-latitude NH. The three definitions are as follows:Definition 1: spring: April-May; summer: June-August; autumn: September-OctoberDefinition 2: spring: April-May; summer: June-August; autumn: SeptemberDefinition 3: spring: May; summer: June-August; autumn: September-OctoberWe then performed the same analyses for the three different season definitions. The results of Definition 1 are presented in the main text, and the results of the other two definitions are presented in the How to cite this article: Wu, X. et al. Seasonal divergence in the interannual responses of Northern Hemisphere vegetation activity to variations in diurnal climate. Sci. Rep. 6, 19000; doi: 10.1038/srep19000 (2016).


Aims. To investigate the impact of blood type, functional polymorphism (T-1676C) of the COX-1 gene promoter, and clinical factors on the development of peptic ulcer during cardiovascular prophylaxis with low-dose aspirin. Methods. In a case-control study including 111 low-dose aspirin users with peptic ulcers and 109 controls (asymptomatic aspirin users), the polymorphism (T-1676C) of the COX-1 gene promoter was genotyped, and blood type, H pylori status, and clinical factors were assessed. Results. Univariate analysis showed no significant differences in genotype frequencies of the COX-1 gene at position -1676 between the peptic ulcer group and control group. Multivariate analysis revealed that blood type O, advanced age, history of peptic ulcer, and concomitant use of NSAID were the independent risk factors for the development of peptic ulcer with the odds ratios of the 2.1, 3.1, 27.6, and 2.9, respectively. Conclusion. The C-1676T polymorphism in the COX-1 gene promoter is not a risk factor for ulcer formation during treatment with low-dose aspirin. Blood type O, advanced age, history of peptic ulcer, and concomitant use of NSAID are of independent significance in predicting peptic ulcer development during treatment with low-dose aspirin. Low-dose aspirin 75–325 mg/day) is widely used in the prevention of myocardial infarction or ischemic stroke . Besides5 mg/day  Helicobacter pylori (H. pylori) infection, and history of alcohol abuse were risk factors for gastroduodenal ulcer in low-dose aspirin users [Previous studies revealed that a history of ulcer, advanced age >70 years), concomitant use of nonsteroidal anti-inflammatory drugs (NSAIDs), use of dual antiplatelet therapy,in users , 7. Howe0 years, in users . Nonethe COX-1 gene promoter has been identified and was reported to alter putative transcription factor  binding sites [ COX-1 gene promoter was a significant risk factor for the NSAID-induced ulcer in Japan [ COX-1 genetic polymorphism also plays an important role in the development of peptic ulcer in low-dose aspirin users remains unclear.Cyclooxygenase-1 (COX-1) is a constitutively expressed enzyme that generates prostaglandins for gastrointestinal integrity. Aspirin can inhibit COX-1 enzyme and decrease prostaglandin synthesis of gastrointestinal tract. Recently, functional polymorphism (T-1676C) in theng sites , 10. Ariin Japan . HoweverThe aim of this case-control study was to investigate the clinical risk factors and genetic markers for the development of peptic ulcer during cardiovascular prophylaxis with low-dose aspirin.A total of 111 consecutive low-dose 75–325 mg/day) aspirin users with peptic ulcer, who attended the Kaohsiung Veterans General Hospital or Kaohsiung Medical University Hospital, were included in this study. The reason for choosing this dosage range of aspirin is because the common dose of aspirin used for the prevention of cardiovascular diseases varies between 75 and 325 mg daily [25 mg/day H. pylori infection was based on the result of rapid urease test. An ulcer was defined as a circumscribed mucosal break 3 mm or more in diameter, with a well-defined ulcer crater in the stomach or duodenum [Endoscopies were performed with the Olympus GIF XV10 and GIF XQ200 . During endoscopy, biopsies over antrum and body were performed for rapid urease test (with one specimen from the antrum and another one from the body). The rapid urease test was performed according to our previous studies . Rapid uduodenum . The siz COX-1 genotyping. To assess the significance of clinical characteristics, the following data were recorded for each patient: age, sex, blood type, smoking, alcohol consumption, coffee consumption, tea consumption, previous history of peptic ulcer, and use of thienopyridine, coumadin, steroid, or NSAIDs within 2 weeks prior to endoscopy. Coffee or tea consumption was defined as drinking 1 cup or more per day.Before endoscopy, venous blood was drawn for COX-1 genotype (T-1676C) was determined using the restriction fragment length polymorphism (RFLP). The primer set  [2 , 2.5 mM each of the 4 deoxynucleotide triphosphates , 0.5 M of each amplification primer, and 40–100 ng of genomic DNA mixed up to a final volume of 25 μL. The PCR analyses were run on a PCR thermal cycler  with the following profile: 5 min of denaturation at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 64°C, and 60 s at 72°C, with a final elongation at 72°C for 7 min. The amplified DNA fragments (243 base pairs) contained two TspR I restriction sites. After TspR I digestion, fragments with172 base pairs and 71 base pairs were observed for genotype TT; fragments with 172 base pairs, 88 base pairs, 84 base pairs, and 71 base pairs were observed for genotype TC; and fragments with 88 base pairs, 84 base pairs, and 71 base pairs were observed for genotype CC. Then the PCR products were digested by TspR I restriction enzymes in a reaction mixture (20 μL) containing PCR products (15 μL), 1X Buffer 4, 1X BSA, and TspR I (0.5 U/μL) at 65°C for 16 hours. The digested fragments were separated and analyzed by electrophoresis using 3% agarose gel. Genotypes of COX-1 gene promoter were classified into the following three groups: T/T, T/C, and C/C.Genomic DNA was extracted from 3 mL of whole blood by the use of a QIAamp DNA Extraction Mini Kit . TheACCT-3′)  was used t-tests were used to compare the mean values of the variables considered continuous in the peptic ulcer patients and controls. The chi-square test with or without Yate's correction for continuity and Fisher's exact test when appropriate were applied to analyze the categorized variables. Differences were considered to be significant at P < 0.05. A stepwise forward conditional method was used to identify the independent risk factors for the development of peptic ulcer in low-dose aspirin users. The studied variables included the following: age (<60 or ≥60 years), sex, blood type (O type or non-O type), history of smoking (<1 pack/week or ≥1 pack/week), history of alcohol consumption (<80 g/day or ≥80 g/day), H. pylori status (presence or absence), and genotype of COX-1 gene promoter.Statistical evaluations were performed using the SPSS/Windows computer software package . Two-sample COX-1 gene promoter in healthy subjects was 47%. We estimated that a 20% difference in the allele frequency could be present in low-dose aspirin users with peptic ulcer. Based on this assumption, 93 subjects had to be studied in each group to yield a statistical power of 0.80 and an α value of 0.05.According to a previous study , the fren = 90; duodenal ulcer: n = 10; both gastric and duodenal ulcers: n = 11) and 114 controls. P = 0.001). In addition, the rates of peptic ulcer history and the use of NSAID of peptic ulcer subjects were significantly higher than the controls . However, the peptic ulcer group had a lower rate of coffee drinking than the control group . H. pylori infection was detected in 26.2% of peptic ulcer subjects and in 47.5% of the controls. The infection rate was significantly lower in the peptic ulcer group than in the control group (P = 0.005). The two groups were similar with respect to gender, tea consumption, alcohol consumption, dual antiplatelet therapy with thienopyridine, and use of steroids.Two hundred and twenty aspirin users  were recruited for the study. They included 111 subjects with peptic ulcer disease (gastric ulcer:  COX-1 gene promoter in aspirin users with and without peptic ulcer. The distributions of blood groups between aspirin users with and without peptic ulcer were different (P = 0.047). Peptic ulcer group had a higher rate of blood type O than the control group . The T/T, C/T, and C/C genotypes at position -1676 of the COX-1 gene promoter in aspirin users with peptic ulcer were 24.7%, 66.3%, and 9.0%, respectively. No significant differences in genotype frequencies of the COX-1 gene were found between the peptic ulcer group and control group (P = 0.245).A stepwise forward logistic regression analysis for clinical and genetic variables was performed to search for the independent risk factors of peptic ulcer development in low-dose aspirin users. Multivariate analysis indicated that the advanced age, history of peptic ulcer, use of NSAID, and blood type O were the independent risk factors for the development of peptic ulcer . The odd COX-1 gene promoter is not a risk factor for ulcer formation during treatment with low-dose aspirin.Aspirin, usually available as an over-the-counter drug, is one of the most used drugs worldwide. In Western countries, 6 to 12% of the general population is exposed to low-dose aspirin , 16. It Blood group O has traditionally been associated with the risk of developing peptic ulcer , 19. How COX gene polymorphisms could be important in the pathogenesis of peptic ulcer disease because the function of cyclooxygenases affects prostaglandin formation and its protective effect at the level of the gastric mucosa. A previous study showed that the A-842G polymorphism did not play a significant role in the development of ulcer in NSAID users in the Japanese population [ COX-1 gene promoter and peptic ulcer development in low-dose aspirin users. Our data indicated that no significant differences in genotype frequencies of the C-1676T polymorphism in the COX-1 gene promoter were found between the peptic ulcer group and control group. However, Arisawa et al. reported that the C-1676T functional polymorphism in the COX-1 gene promoter was related to the development of NSAID-induced ulcer [Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandins. As a corollary,pulation . In thised ulcer . The reaIn this study, we confirmed that advanced age, history of peptic ulcer, and use of NSAID were independent factors predictive of ulcer development in low-dose aspirin users. History of peptic ulcer is the most important clinical risk factor for ulcer development in low-dose aspirin use with an odds ratio of 27.6. The finding was consistent with previous studies that also identified a history of peptic ulcer as a clinical risk factor for peptic ulcer in low-dose aspirin users , 21. PPISeveral other independent studies , 27 have H. pylori infection was detected in 26.2% of peptic ulcer subjects and in 47.5% of the controls. The infection rate was significantly lower in the peptic ulcer group than in the control group. The reason for the lower H. pylori infection rate in the peptic ulcer group was probably due to prior anti-H. pylori therapy in many aspirin users in this patient group. Among the patients in peptic ulcer group, 50.5% of the subjects had a history of prior peptic ulcer. Multivariate analysis indicated that the history of peptic ulcer was the independent risk factor for the development of peptic ulcer and H. pylori status was removed from independent factors related to peptic ulcer development following logistic regression analysis.In the current study,The strengths of this study included investigating both clinical and genetic risk factors for ulcer development in low-dose aspirin users and prospective assessment of clinical data of the recruited subjects. However, it has several limitations. First, the sample size was moderate and possibly unable to detect minor independent risk factors for the development of peptic ulcer in low-dose aspirin users. Secondly, aspirin users who took PPIs or histamine-2 receptor antagonists before endoscopy were excluded. We therefore could not identify some protective factors for ulcer development in low-dose aspirin users. Thirdly, it was a case-control study and selection bias of recruited cases could not be completely ruled out. A large-scale, long-term cohort study is therefore merited to clarify the risk factors identified in this work. COX-1 gene promoter is not a risk factor for ulcer formation during treatment with low-dose aspirin.In conclusion, blood type O, advanced age, history of peptic ulcer, and use of nonaspirin NSAID are independent risk factors for development of peptic ulcer in low-dose aspirin users. The C-1676T polymorphism in the


The mechanisms eliciting colorectal adenocarcinoma are not well understood and the FBXL20 gene is problematic as it exhibits an abnormal expression in colorectal cancer cells. In the present study a recombinant plasmid, pReceiver-M03-FBL20 expression plasmid was constructed, which overexpressed FBXL20; this was transfected into Lovo cells to form Lovo-FBL20 cells. The FBXL20 expression level was examined by quantitative polymerase chain reaction (qPCR) and western blot analysis. The cell viability and invasion capacity were measured using cell counting kit 8, Transwell chamber and wound healing assays, respectively. The associated genes, including E-cadherin, β-catenin, c-Myc, SET nuclear oncogene, protein phosphatase-2A, Axin, p53 and caspase 3, were detected by qPCR and western blotting. It was demonstrated that the FBXL20 expression level was markedly upregulated in the Lovo-FBL20 cells transfected with pReceiver-M03-FBL20 expression plasmid, compared with that of the Lovo cells. In addition, the cell viability and invasion capacity of the Lovo-FBL20 cells were significantly increased. These increases correlated with a significant upregulation in the expression level of β-catenin and c-Myc, and a downregulated expression level of E-cadherin. The results of the present study indicate that FBXL20 may mediate the ubiquitin degradation of E-cadherin resulting in an increased invasive ability of malignant cells. Colorectal adenocarcinoma is one of the most common types of malignant tumor, with an increasing prevalence over recent years. It has the second highest incidence rate of all types of cancers and it is the second most common cause of cancer-associated mortality in Europe ,2. Carciet al , with a 1,308-bp open reading frame. It is located on the human chromosome 17q21.2, and predicts to encode a 436-amino-acid protein that contains an F-box motif, which is the key feature of F-box proteins (FBPs). The essential role of FBPs has been demonstrated in studies of various types of species for ubiquitin-mediated degradation of cellular regulatory proteins. Welcker et al  were purchased from the American Type Culture Collection . The colorectal cancer (CRC) cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum.BamH1/BbsI site of the pReceiver-M03 expression plasmid. The engineered pReceiver-M03-FBL20 expression plasmid was verified by restriction digestion and sequenced by the Beijing Genomic Institute . The recombinant pReceiver-M03-negative control (NC) vector was obtained as described above, however, the inserted sequence was not homologous with any human gene. The Lovo cells were transfected with 1 μg pReceiver-M03-FBL20 expression plasmid according to the manufacturer’s instructions .The oligonucleotides containing the whole coding DNA sequences of the FBXL20 gene were synthesized through a chemical method . The annealed complementary oligonucleotides were subsequently inserted into the Total RNA was extracted from the colorectal adenocarcinoma cells by the TRIzol reagent (Invitrogen Life Technologies) and purified with RNeasy columns . An RNase-Free DNase kit  was used to digest DNA. Cells were lysed with radioimmunoprecipitation assay buffer  containing protease inhibitors  and protein quantification was conducted using a bicinchoninic acid protein assay kit (Beyotime Co.).The proliferation assay was performed 24 h after transfecting the pReceiver-M03-FBL20 expression plasmid into a 96-well plate by the addition of 10 μl cell counting kit 8  into the medium, followed by incubation for 30 min under normal cell culture conditions. Cell viability was measured by absorbance at 450 nm using a microplate reader .2 atmosphere), the cells in the upper surface of the membrane were removed using a cotton swab and the cells that had migrated to the lower surface of the membrane were fixed and stained. The migrated cells on the lower surface of the membrane filter were scored from six random fields under a CKX31 microscope . The results are shown as percentage migrating rate (%): % =  × 100. The wound healing assay was performed according to the manufacturer’s instructions. To generate a wound field, cells were cultured until they formed a monolayer around the insert. Subsequent to removing the insert, an open wound field was generated and cells were able to migrate from either side of the gap.Cell invasion assays were performed with the 24-well cell invasion assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Cells (n=900) were resuspended in culture medium without FBS and placed in the upper Transwell chamber in triplicate. After a 24-h incubation at 37°C  using the SYBR Premix Ex Taq mix (Takara) according to the manufacturer’s instructions. The primers were purchased from Sangon Biotech. GAPDH served as an endogenous control.qPCR was performed in triplicate with a MastercyclerThe total cell protein (50 μg) was used for western blotting. The samples were resolved in 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were immersed in western blocking buffer (Beyotime Co.) for 1 h and probed with the primary polyclonal antibody against FBL20, E-cadherin, SET, Axin, PP2A, p53, caspase 3 and β-actin  overnight at 4°C. The blots were washed in tris-buffered saline containing 0.1% Tween-20 and labeled with horseradish peroxidase-conjugated secondary anti-rabbit antibody . Proteins were enhanced by BeyoECL Plus (Beyotime Co.) for visualization. The protein expression levels were expressed relative to β-actin levels.The results presented in the current study are the means ± standard error of the mean. Statistical analysis was performed by Student’s t-test, Fisher’s exact probability test and analysis of variance with SPSS 17.0 software . P<0.05 was considered to indicate a statistically significant difference.FBXL20 expression levels in the CRC cell lines (five types) were identified by qPCR. It was shown that the FBXL20 expression level was significantly lower in the Lovo cell line compared with the SW480, SW620, Ls174T, HT29 and HCT116 cell lines .To investigate the function of FBXL20, the pReceiver-M03-FBL20 expression plasmid, which overexpressed the FBXL20 gene, was transfected into the Lovo cell line. At 48 h after transfecting the pReceiver-M03-FBL20 expression plasmid or pReceiver-M03-NC into the Lovo cells, the FBXL20 expression level was verified by qPCR  and western blotting , compared with that in the Lovo cells .Cell viability (CCK8 assay) and cell invasion  were performed using the Lovo cells that were transiently transfected with the pReceiver-M03-FBL20 expression plasmid. It was observed that the cell proliferation and migration activity was significantly increased in the Lovo-FBL20 cells compared with that of the Lovo cells. The percentages for the migrating rates in Lovo-FBL20 and Lovo cells were 42.18% (37.9±85.28%) and 14.05% (12.6±16.05%), respectively (data not shown). The difference between the Lovo-FBL20 and Lovo cells was identified to be statistically significant P=0.007;  and 3.To investigate the function of FBXL20, specific molecules were detected in the Lovo-FBL20 cells by qPCR and western blotting. qPCR and western blotting demonstrated that the mRNA expression and protein expression level of E-cadherin in the Lovo-FBL20 cells was significantly reduced compared with that in the Lovo cells, and the reduction rates were 63.50 and 90.20%, for mRNA and protein expression, respectively, which was considered to be statistically significant (P=0.001 and P<0.001; The mRNA levels of β-catenin and c-Myc in the Lovo-FBL20 cells were significantly upregulated compared with the Lovo cells. The β-catenin and c-Myc expression levels in the Lovo-FBL20 cells were 2.870 and 3.997-fold, respectively, compared with that in the Lovo cells (P<0.001 and P<0.001; However, the protein expression levels of SET, PP2A and Axin were not found to be significantly different between the Lovo-FBL20 and Lovo cells by western blot analysis .et al (CRC remains a predominant cause of cancer mortality worldwide. Numerous studies have documented the significant roles of the F-Box family in the initiation of CRC. Grim et al  identifiet al . In addiet al (In the present study, cell proliferation activity was significantly increased in the Lovo-FBL20 cells, compared with that of the corresponding control cells. The selective ubiquitination of proteins by ubiquitin E3 ligases exhibits a significant regulatory role in the control of cell differentiation, growth and transformation, and the dysregulation of ubiquitin E3 ligases is often associated with pathological outcomes, including tumorigenesis . Cell pret al  reportedet al . Based oet al (The mechanism by which the upregulated FBXL20 expression level leads to the increased proliferation of the colorectal adenocarcinoma cells remains unknown. The c-Myc and β-catenin expression levels in the present study were significantly upregulated in the Lovo-FBL20 cells compared with those in the corresponding control cells. Numerous studies have confirmed that the development of CRC was closely correlated with the activation of the Wnt signaling pathway ,16,17. Fin vivo (A notable finding of the present study is that the E-cadherin expression level was significantly reduced and the migration ability of the Lovo-FFBL20 cells was significantly increased, which indicated that the migration ability was correlated with the E-cadherin expression level. These data also support the recent observation that downregulation of E-cadherin in pancreatic adenocarcinoma may promote invasion and metastasis of cancer cells . In addiin vivo . Recent in vivo . Cell adin vivo .et al (P53 is a well-known tumor suppressor gene whose loss of function emphasizes the most common genetic alteration in human malignancy. It is involved in DNA damage repair, cell cycle regulation, apoptosis, ageing and cellular senescence . In the et al  reportedet al . In addi-subunit serine/threonine phosphatase that is integral for intracellular signaling, cell cycle progression and gene regulation. Ikeda et al (In the present study, SET expression was not found to be correlated with an increased FBXL20 expression in the Lovo-FBL20 cells. Although, it was shown that the SET expression level was markedly increased following silencing of the FBXL20 gene in the colorectal adenocarcinoma cell lines, SW480 and SW620. These findings support the results that the Axin level did not increase with the upregulation of p53 in the Lovo-FBL20 cells. PP2A is a multida et al  reportedda et al . NotablyIn conclusion, the present data on the roles of FBXL20 in colorectal adenocarcinoma cells are comparable with other F-Box family members, which mediate the specific substrate protein into the ubiquitin proteasome pathway. The present study indicated that FBXL20 may mediate the ubiquitin degradation of E-cadherin resulting in an increased invasive ability of malignant cells. However, further studies are required to identify all of the factors that are involved in the FBXL20-mediated ubiquitin proteasome process in colorectal adenocarcinoma.